• Collagen 4 binds to the VWF A1 domain, and this binding is reduced or abrogated by select VWF A1 domain sequence variations.• Platelet binding to collagen 4 under flow conditions is dependent on the presence of VWF.Von Willebrand factor (VWF) contains binding sites for platelets and for vascular collagens to facilitate clot formation at sites of injury. Although previous work has shown that VWF can bind type IV collagen (collagen 4), little characterization of this interaction has been performed. We examined the binding of VWF to collagen 4 in vitro and extended this characterization to a murine model of defective VWF-collagen 4 interactions. The interactions of VWF and collagen 4 were further studied using plasma samples from a large study of both healthy controls and subjects with different types of von Willebrand disease (VWD). Our results show that collagen 4 appears to bind VWF exclusively via the VWF A1 domain, and that specific sequence variations identified through VWF patient samples and through site-directed mutagenesis in the VWF A1 domain can decrease or abrogate this interaction. In addition, VWF-dependent platelet binding to collagen 4 under flow conditions requires an intact VWF A1 domain. We observed that decreased binding to collagen 4 was associated with select VWF A1 domain sequence variations in type 1 and type 2M VWD. This suggests an additional mechanism through which VWF variants may alter hemostasis.
ABSTRACT. The 2-deo~y-['~C]-glucose (2-DG) method of Sokoloff was used to assess regional cerebral glucose utilization (CGU) in the immature rat. The 7-d postnatal rats received 2.5 pCi 2-DG subcutaneously, after which blood was collected for measurement of plasma glucose and 2-DG activity at intervals up to 90 min. The brains of the 90-min rat pups either were frozen for analysis of glucose concentration and chromatographic separation of 2-DG and 2-DG-6-phosphate or for ['4C]-autoradiography. A lumped constant of 0.55 was calculated from plasma and brain glucose levels of 6.4 and 1.62 mmol/L.kg, respectively. Of the [I4C] activity in brain, 75.6% was in the 2-DG-6-phosphate fraction; this percent was substituted for Kl", K2*, and K3" in the Sokoloff equation. Cerebral hemispheric CGU (n = 6) averaged 11.4 + 1.5 wmo1/100 g/min, 1/10 the value of adult rat brain. Rates in 16 brain structures (n = 10) ranged from 7.8 (frontal white matter) to 16.9 (cerebellum) pmo1/100 g/min. During hypoxiaischemia (unilateral common carotid artery ligation combined with exposure to 8% oxygen), the lumped constant increased to 1.04, and 99% of 2-DG was converted to 2-DG-6-phosphate. Increases in CGU occurred in all eight structures of the cerebral hemisphere ipsilateral to the carotid artery occlusion (n = 9), ranging from 287% (frontal white matter) to 445% (striatum) of control values (p < 0.05). Relatively comparable elevations in CGU (234-435% of control) occurred in the contralateral cerebral hemisphere, which were not significantly different from those of the ipsilateral hemisphere. The relatively proportionate increases in regional CGU of the two cerebral hemispheres, only one of which sustains tissue injury, suggest interhemispheric differences in the extent to which glucose is metabolized via anaerobic glycolysis to maintain cellular energy production. The investigation demonstrates the feasibility of measuring regional CGU in the small laboratory animal, which is applicable to a variety of physiologic and pathologic situations. (Pediatr Res 26: 208-214,1989) Abbreviations CGU, cerebral glucose utilization rCGU, regional CGU 2-DG, 2-deoxyglucose 2-DG-6-P, 2-deoxyglucose-6-phosphate s.c., ~ubcutaneous LC, lumped constant -P, high-energy phosphate bond The 2-DG technique, as originally developed by Sokoloff et al. (I), has become an established procedure to measure rCGU in adult animals. The method has been used in numerous species under a variety of physiologic and pathologic situations (2). Furthermore, the method provides the theoretical and practical basis for the measurement of rCGU in humans, including infants and children, using positron emission tomography (3, 4). Although rCGU has been determined in large perinatal animals (5-7), a systematic investigation of its applicability to the small laboratory animal has yet to be accomplished. We here describe a feasibility study to measure rCGU, using 2-DG, in the immature rat; and we have used the technique to ascertain the nature and extent of alterations in regional...
Administration of mobilized peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy rapidly restores multilineage hematopoiesis, but the ability of such products to restore lymphocyte populations remains unclear. In this report, we evaluated immune reconstitution in a series of patients treated with sequential cycles of high-dose chemotherapy, followed by autologous PBPC infusions (median CD34+ cell dose 7.2 × 106 cells/kg [range 2-29.3]). Although patients experienced rapid reconstitution of B cells and CD8+ T cells, we observed CD4 depletion and diminished immune responsiveness in all patients for several months after completion of therapy. Mature CD4+ T cells contained within the grafts did not appear to contribute substantially to immune reconstitution because CD4 counts did not differ between recipients of unmanipulated T-cell replete infusions versus CD34 selected, T-cell–depleted infusions. Rather, at 12 months after therapy, total CD4 count was inversely proportional to age (ρ = −0.78,P = .04), but showed no relationship to CD34 cell dose (ρ = −0.42, P = .26), suggesting that age-related changes within the host are largely responsible for the limited immune reconstitution observed. These results demonstrate that in the autologous setting, the infusion of large numbers of PBPCs is not sufficient to restore T-cell immune competence and emphasize that specific approaches to enhance immune reconstitution are necessary if immune-based therapy is to be used to eradicate minimal residual disease after autologous PBPC transplantation.
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